The present invention relates to the protection of cells that would otherwise die as a result of stroke or an ischemic event.
Ischemia is an acute condition associated with an inadequate flow of oxygenated blood to a part of the body, caused by the constriction or blockage of the blood vessels supplying it. Ischemia occurs any time that blood flow to a tissue is reduced below a critical level. This reduction in blood flow can result from: (i) the blockage of a vessel by an embolus (blood clot); (ii) the blockage of a vessel due to atherosclerosis; (iii) the breakage of a blood vessel (a bleeding stroke); (iv) the blockage of a blood vessel due to vasoconstriction such as occurs during vasospasms and possibly, during transient ischemic attacks (TIA) and following subarachnoid hemorrhage. Conditions in which ischemia occurs further include (i) myocardial infarction; (ii) trauma; and (iii) during cardiac and thoracic surgery and neurosurgery (blood flow needs to be reduced or stopped to achieve the aims of surgery). During myocardial infarct, stoppage of the heart or damage occurs which reduces the flow of blood to organs, and ischemia results. Cardiac tissue itself is also subjected to ischemic damage. During various surgeries, reduction of blood flow, clots or air bubbles generated can lead to significant ischemic damage.
When an ischemic event occurs, there is a gradation of injury that arises from the ischemic site. The cells at the site of blood flow restriction, undergo necrosis and form the core of a lesion. A penumbra is formed around the core where the injury is not immediately fatal but progresses slowly toward cell death. This progression to cell death may be reversed upon reestablishment of blood flow within a short time of the ischemic event.
Focal ischemia encompasses cerebrovascular disease (stroke), subarachnoid hemorrhage (SAH) and trauma Stroke is the third leading cause of morbidity in the United States, with over 500,000 cases per year, including 150,000 deaths annually. Post-stroke sequelae are mortality and debilitating chronic neurological complications which result from neuronal damage for which prevention or treatment are not currently available.
Following a stroke, the core area shows signs of cell death, but cells in the penumbra remain alive for a period of time although malfunctioning and will, in several days, resemble the necrotic core. The neurons in the penumbra seem to malfunction in a graded manner with respect to regional blood flow. As the blood flow is depleted, neurons fall electrically silent, their ionic gradients decay, the cells depolarize and then they die. Endothelial cells of the brain capillaries undergo swelling and the luminal diameter of the capillaries decrease. Associated with these events, the blood brain barrier appears to be disrupted, and an inflammatory response follows which further interrupts blood flow and the access of cells to oxygen.
The effects of a stroke on neurons result from the depletion of energy sources associated with oxygen deprivation which in turn disrupts the critically important ion pumps responsible for electrical signaling and neurotransmitter release. The failure of the ATP-dependant ion specific pumps to maintain ion gradients through active transport of sodium, chlorine, hydrogen, and calcium ions out of the cell and potassium ions into the cell results in a series of adverse biochemical events. For example, increase in intracellular calcium ion levels results in: (I) the production of free radicals that extensively damage lipids and proteins; (ii) the disruption of calcium sensitive receptors such as the N-methyl D-aspartate (NMDA) and the xcex1-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) synaptic glutamate receptors; (iii) the swelling of cells with water as a result of abnormal accumulation of ions; and (iv) the decrease in intracellular pH. The alteration in metabolism within the cell further results in the accumulation of ions in the cells as energy sources are depleted. For example, anaerobic glycolysis that forms lactic acid, replaces the normal aerobic glycolysis pathways in the mitochondria. This results in acidosis that results in further accumulation of calcium ions in the cell.
Despite the frequency of occurrence of ischemia (including stroke) and despite the serious nature of the outcome for the patient, treatments for these conditions have proven to be elusive. There are two basic approaches that have been undertaken to rescue degenerating cells in the penumbra. The first and most effective approach to date has been the identification of blood clot dissolvers that bring about rapid removal of the vascular blockage that restricts blood flow to the cells. Recombinant tissue plasminogen activator (TPA) has been approved by the Federal Drug Administration for use in dissolving clots that cause ischemia in thrombotic stroke. Nevertheless, adverse side effects are associated with the use of TPA. For example, a consequence of the breakdown of blood clots by TPA treatment is cerebral hemorrhaging that results from blood vessel damage caused by the ischemia. A second basic approach to treating degenerating cells deprived of oxygen is to protect the cells from damage that accumulates from the associated energy deficit. To this end, glutamate antagonists and calcium channel antagonists have been most thoroughly investigated. None of these have proven to be substantially efficacious but they are still in early clinical development. The pathophysiology and treatment of focal cerebral ischemia has been reviewed by B. K. Seisjo, J. Neurosurgery, 1992, vol. 77, p. 169-184 and 337-354.
In addition to the targets of drug development described by Seisjo (1992), epidemiological studies have shown that women undergoing hormone replacement therapy with estrogen and progesterone experienced a reduction in the incidence and severity of heart disease. This correlation was further investigated for stroke with mixed results. A 10-year epidemiological study on 48,000 women reported by Stampfer et al. (New England Journal of Medicine, 1991, vol. 325, p. 756) concluded that there was a correlation between use of estrogen and decrease in incidence of coronary heart disease, but no decrease in the incidence of stroke was observed. In contrast, a report by Wren (The Medical Journal of Australia, 1992, vol. 157, p. 204) who reviewed 100 articles directed to the question as to whether estrogens reduce the risk of atherosclerosis and myocardial infarction, concluded that estrogens in hormone replacement therapies significantly reduce the incidence of myocardial infarction and stroke and may accomplish this at the site of the blood vessel wall. This conclusion was further supported by Falkeborn et al. Arch Intern. Med., 1993, vol. 153, p. 1201. The above correlation between estrogen replacement therapy and reduced incidence of stroke relies on epidemiological data only. No biochemical data were analyzed to interpret or support these conclusions, nor is there any information as to reduction in ischemic lesion or morbidity with hormone use. Furthermore, these studies were restricted to the patients receiving long-term hormone replacement treatment. No studies were performed on patients who might be administered estrogen therapeutically shortly before, during, or after a stroke for the first time. Furthermore, the studies were limited to estrogens utilized in estrogen replacement therapy. No studies were performed on any non-sex related estrogens that might be used in treating males or females.
Studies have been conducted on the neuroprotective effects of steroids in which glucocorticosteroid for example was found to have a positive effect in reducing spinal cord injury but had a negative effect on hippocampal neurodegeneration. For example, Hall (J. Neurosurg vol. 76, 13-22 (1992)) noted that the glucocorticoid steroid, methylprednisolone, believed to involve the inhibition of oxygen free radical-induced lipid peroxidation, could improve the 6-month recovery of patients with spinal cord injury when administered in an intensive 24hour intravenous regimen beginning within 8 hours after injury. However, when the steroid was examined for selective protection of neuronal necrosis of hippocampal neurons, it was found that the hippocampal neuronal loss was significantly worsened by glucocorticoid steroid dosing suggesting that this hormone is unsuitable for treating acute cerebral ischemic. Hall reported that substitution of a complex amine on a non-glucocorticoid steroid in place of the 21xe2x80x2-hydroxyl functionality results in an enhancement of lipid anti-oxidant activity. No data were provided concerning the behavior of this molecule in treating ischemic events or in neuroprotection of neurons in the brain. Additionally, free radical scavenging activity has been reported for a lazaroid, another non-glucocorticoid steroid having a substituted 21xe2x80x2-hydroxyl functionality, but there is no evidence that this compound is significantly efficacious for treating stroke or other forms of ischemia.
The invention satisfies the above need. Novel methods are provided for prevention and treatment of ischemic damage using estrogen compounds.
A preferred embodiment of the invention provides a method for conferring protection on a population of cells associated with an ischemic focus, in a subject following an ischemic event that includes the steps of providing subcutaneously an estrogen compound in a drug delivery system in which the estrogen compound is dissolved in oil with or without additional excipients such as solvents, stabilizers or preservatives, so as to confer protection on the population of cells. Further embodiments include selecting a proximate time for administering the effective dose of the estrogen compound that is prior to the ischemic event. Alternatively, the estrogen compound may be administered within an effective proximate time after the ischemic event. The method of the invention may be applied to any of a cerebrovascular disease, subarachnoid hemorrhage, myocardial infarct, surgery, and trauma. In particular, when the ischemic event is a stroke, the protected cells include at least one of neurons and endothelial cells.
The method utilizes an estrogen compound that may include alpha isomers or beta isomers of estrogen compounds. Examples of different isomers are provided wherein the estrogen compound is selected from the group consisting of 17xcex1-estradiol and 17xcex2-estradiol.
In a preferred embodiment of the invention, a method is provided for protecting cells in a subject from degeneration during or after an ischemic event. The steps of the method include identifying a susceptible subject, providing an effective dose of an estrogen compound prior to or after the ischemic event, and protecting cells from degeneration otherwise occurring in the absence of the estrogen compound.
In a further embodiment of the invention, a method is provided for treating stroke in a subject, including the steps of providing an effective dose of an estrogen compound in a pharmaceutical formulation and administering the formulation to the subject so as to reduce the adverse effects of the stroke.
The invention in another embodiment provides a method for conferring protection on a population of cells associated with ischemia, in a subject following an ischemic event, comprising: (a) providing an estrogen compound formulated in an oil vehicle; and (b) administering an effective amount of the compound over a course that includes at least one dose within a time that is effectively proximate to the ischemic event, so as to confer protection on the population of cells. Further in this embodiment in (b) the estrogen compound is administered by subcutaneous injection.
In another embodiment, the invention provides a method of synthesis of ent-17xcex2-estradiol from [3R-(3xcex1,3axcex1,9axcex1,9bxcex2)]-3-(1,1-dimethylethoxy)-1,2,3,3a,4,5,8,9,9a,9b-decahydro-3a-methyl-6-[2-(2-methyl-1,3-dioxolan-2-yl)ethyl-7H-benz[e]inden-7-one, comprising: reducing the double bond of [3R-(3xcex1,3axcex1,9axcex1,9bxcex2)]-3-(1,1-dimethylethoxy)-1,2,3,3a,4,5,8,9,9a,9b-decahydro-3a-methyl-6-[2-(2-methyl-1,3-dioxolan-2-yl)ethyl-7H-benz[e]inden-7-one to obtain tricyclic compound [3R-(3xcex1,3axcex1,5axcex2,6xcex2,9axcex1,9bxcex2)]-3-(1,1-dimethylethoxy)-dodecahydro-3a-methyl-6-[2-(2-methyl-1,3-dioxolan-2-yl)ethyl-7H-benz[e]inden-7-one; cyclizing the tricyclic compound [3R-(3xcex1,3axcex1,5axcex2,6xcex2,9axcex1,9bxcex2)]-3-(1,1-dimethylethoxy)-dodecahydro-3a-methyl-6-[2-(2-methyl-1,3-dioxolan-2-yl)ethyl-7H-benz[e]inden-7-one to obtain ent-19-nortestosterone; esterifying the hydroxy group of ent-19-nortestosterone to obtain ent-19-nortestosterone,17-acetate; aromatizing the steroid A ring of ent-19-nortestosterone,17-acetate to obtain ent-17xcex2-estradiol,17-acetate; and saponifying ent-17xcex2-estradiol,17-acetate to remove the 17-acetate group, to obtain ent-17xcex2-estradiol.
According to this method, reducing the double bond of [3R-(3xcex1,3axcex1,9axcex1,9bxcex2)]-3-(1,1-dimethylethoxy)-1,2,3,3a,4,5,8,9,9a,9b-decahydro-3a-methyl-6-[2-(2-methyl-1,3-dioxolan-2-yl)ethyl-7H-benz[e]inden-7-one is obtained by a step selected from the group consisting of: using lithium in liquid ammonia, and using catalytic hydrogenation.
Another embodiment of the invention provides the compound [3R-(3xcex1,3axcex1,5axcex2,6xcex2,9axcex1,9bxcex2)]-3-(1,1-dimethylethoxy)-dodecahydro-3a-methyl-6-[2-(2-methyl-1,3-dioxolan-2-yl)ethyl-7H-benz[e]inden-7-one.
A further embodiment of the invention provides the compound ent-17xcex2-estradiol,17-acetate. Yet another embodiment of the invention is the compound ent-19-nortestosterone, 17-acetate.